PROJECT SUMMARY This Small Business Innovation Research (SBIR) Phase I project aims to develop an accurate, rapid, and commercially available microfluidic sensor that measures low molecular weight S-nitrosothiols in biological samples. S-nitrosothiols (RSNOs), the primary transporters of nitric oxide (NO) in physiology, play a critical role for NO's bioactivity. Homeostatic control of low molecular weight RSNOs, especially S-nitrosoglutathione, is lost in many disease states. Such dysfunction is thought to contribute to the underlying disease pathology. Despite the promise of low molecular weight RSNOs as mechanistic biomarkers, no simple-to-use, standardized tools currently exist for measuring these compounds. There are vast disagreements in the literature regarding the reference ranges for low molecular weight RSNOs, complicating the clinical understanding of these species. As a result, RSNOs have been identified as candidate biomarkers for many inflammatory and respiratory diseases. Yet, little work has validated these findings or determined their clinical utility. A standardized, accurate, facile and commercially ready device to measure RSNOs would enable important clinical research. A simple to use device could resolve fundamental questions regarding the formation and degradation of RSNOs in disease, and help further a general understanding of RSNOs' roles in cellular physiology, signaling and apoptosis. To address the gap in acceptable RSNO measurement techniques, we have begun to develop an innovative, straightforward and inexpensive device for measuring RSNOs in small volumes of biological fluids. The device is based on our core technology?a microfluidic NO sensor. Using a visible light emitting diode (LED), the RSNOs are photolytically broken down to NO which is then detected electrochemically. In this format, the signal generated from NO oxidation is proportional to the RSNO content in the original sample. Employing visible light photolysis along with our microfluidic NO sensor presents several key advantages: (1) our microfluidic sensor requires minimal sample volume (~50 L) and reduces interference from other common electroactive species; (2) the use of visible light to cleave RSNOs (as opposed to UV light-mediated strategies developed by others) does not generate contamination from photolytic reduction of endogenous nitrate; and (3) our sensor features two working electrodes, providing us with a tool to determine (and thus remove) the background signal associated with each unique sample matrix. Leveraging our core competencies in NO chemistry and measurement, we have assembled a talented team of analytical chemists and clinical researchers to develop a simple-to-use, inexpensive device that can measure RSNOs in biologic samples. Once developed, this device will enable new clinical research that evaluates S-nitrosothiols as clinical biomarkers for disease.